An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.


ELP-P10 treatment inhibits MCMV growth in cell culture
To test the inhibitory potential of ELP-P10 in an in vivo mouse model for future experiments, we first investigated the inhibitory potential of ELP-P10 against MCMV in cell culture.Since CMV is species-specific, we performed the MCMV experiments in mouse embryonic fibroblasts (MEFs).The inhibitory potential of the protein biopolymer-delivered peptide, ELP-P10 and ELP (control), was determined by pretreating MEFs with ELP-P10 or ELP with a concentration range of 1.5-200 µM or vehicle-treatment for 1 h and then infecting with MCMV at a low MOI of 0.01.Cells were harvested at 3 dpi, and virus yield was measured by counting the number of plaques using plaque assay.The half maximal inhibitory concentration (IC 50 ) of ELP-P10 was calculated to be 23.41 μM (with a 95% confidence interval of 19.31 to 28.31 μM), and 73.24 μM for ELP (with a 95% confidence interval of 61.15-89.42μM) (Fig. 2a, b).To determine the inhibitory potential of ELP and ELP-P10 after a greater viral challenge, MEFs were pretreated with ELP-P10 or ELP control before infecting them with MCMV at an MOI of 3.0.ELP-P10 and the ELP control were tested at ELP-P10's IC 75 of 80 μM for the high MOI experiment.The titers indicate a > ten-fold reduction in virus growth in ELP-P10 treated cells compared to the vehicle treatment group (Fig. 2c).Although ELP-control treatment showed inhibition of virus growth in infected MEFs, the viral titers in ELP-P10 treated cells were significantly lower compared to ELP-control treated cells.
Next, to rule out any cytotoxic effects of ELP-P10 and to determine whether it could protect cells from CMV infection, a cell viability assay was performed on ELP-P10-treated infected and uninfected MEFs along with ELP-control and ganciclovir (GCV), an FDA-approved drug for CMV, as an appropriate inhibitory control.Following CMV infection, ELP-P10-treated cells had higher cell viability compared to control groups, indicating that ELP-P10 efficiently protected the MCMV-infected cells from virus-induced lytic cell death (Fig. 3a).Importantly, when administered at equimolar concentrations, ELP-P10 treatment resulted in a more efficient protection of cell viability than ELP control or the comparator antiviral drug GCV.To rule out any direct cytotoxic effects of ELP-P10, ELP, or GCV, cell viability was also determined in uninfected fibroblasts.Viability was not affected by ELP-P10, ELP control, or GCV at the concentrations used in the infection assay (Fig. 3b).To corroborate these findings, a metabolic assay was performed to look at the metabolitc activity of the cells following ELP-P10 and ELP treatment using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS).The results largely mirrored the original results using crystal violet (Supplementary Fig. S1).ELP-P10-treated infected cells had higher cell viability compared with control groups, indicating that ELP-P10 protected the MCMV-infected cells from virus-induced lytic cell death (Supplementary Fig. S1a).There was little effect of any of the treatments in uninfected cells.ELP-P10 treatment led to 30% reduction in the viability of the uninfected cells.Although ELP-P10 reduced metabolic activity in the uninfected cells, it still showed a significant protective effect from MCMV infection (Supplementary Fig. S1b).Altogether, this demonstrates the potent antiviral properties of

ELP conjugation of P10 enhances its bioavailability and accumulation in mouse organs
To test our hypothesis that ELP-conjugation to P10 will enhance its bioavailability, pharmacokinetic and biodistribution analysis of rhodamine-labeled P10 and ELP-P10 was performed to determine their plasma half-life and organ-specific accumulation.Upon subcutaneous injection of rhodamine-labeled P10 or ELP-P10, direct measurement of plasma fluorescence revealed that P10 displayed a rapid clearance from plasma compared to ELP-P10, which exhibited a significantly higher concentration in plasma (Fig. 4a).Notably, while P10 began to clear from plasma after 2 h, ELP-P10 demonstrated a peak concentration at 4 h, followed by a gradual decline.The area under the curve (AUC) for ELP-P10 was found to be 5 times greater than that of P10, indicating significantly higher bioavailability 36 for ELP-P10.Moreover, fitting the clearance phase to a two-compartment pharmacokinetic model revealed that ELP-P10 had a terminal half-life of approximately 6.31 h compared to an approximately 1.9 h half-life for the free peptide.For biodistribution, mice were subcutaneously injected with ELP-P10, P10, or vehicle control (saline), and vital organs were harvested 4 h post-injection.Interestingly, the whole organ ex vivo imaging of harvested organs demonstrated a very high accumulation of ELP-P10 in the kidneys, significant accumulation in the liver, and low levels in the other major organs.The unconjugated P10 peptide, however, accumulated at much lower levels than ELP-P10 in the kidneys and liver (Fig. 4b, c).These results collectively suggest that conjugation of P10 with ELP enhanced its bioavailability and accumulation in the liver and kidneys, suggesting its potential to be used as a therapeutic.Given that CMV is a major opportunistic infection in liver and kidney transplant patients 37 , the accumulation of the therapeutic in these organs suggests its potential to be used in transplant settings to reduce CMV infection.

ELP-P10 treatment leads to a reduction in virus titers in MCMV-infected mouse organs
We further investigated the efficacy of P10 and ELP-P10 in a mouse model of CMV infection.Mice were subcutaneously injected with 1000 nmol/Kg of P10, ELP, ELP-P10, or vehicle (saline) and infected with a high dose (10 6 Pfu total) of MCMV (K181 strain) intraperitoneally, one hour post-treatment.Mice were treated with the same dose of test agents every 24 h (based on pharmacokinetic data) and euthanized on day 4 to harvest the vital organs (liver, kidneys, heart, and spleen) (Fig. 5a).All the mice survived for the duration of the experiment.The infected but vehicle-treated group exhibited the highest percentage loss in the body weight (Fig. 5b).In concert with the loss in body weight, we also observed hunched behavior, ruffled fur, and reduced activity in the vehicle-treated group.In contrast, mice treated with ELP-P10, P10 peptide or ELP lost less weight following MCMV infection and did not show hunched behavior, ruffled fur, or reduced activity.
The vital organs of all animals were harvested at the end of the experiment, and viral titer was determined.These data indicated that a maximum reduction in viral titer resulted from ELP-P10 treatment compared to other treatment groups in all the vital organs (Fig. 5c-f).Viral titer was reduced by 79% in the liver (Fig. 5c), 77% in the kidney (Fig. 5d), 71% in the spleen (Fig. 5e), and 90% in the heart (Fig. 5f) following ELP-P10 treatment.Interestingly, ELP control showed significant but less potent reduction of viral titer in the liver, spleen, and heart, and a trend for reduction of titer in the kidney, which was consistent with earlier in vitro experiments demonstrating antiviral activity of the ELP carrier itself.The unconjugated P10 showed a significant reduction in virus titer compared to the control (PBS) treatment in the kidney, heart, and spleen, and a minor reduction in  the liver that did not reach statistical significance.Overall, our results show that ELP-P10 has enhanced efficacy compared to P10 and ELP control.These findings suggest that the conjugation of P10 with ELP enhanced its efficacy to reduce viral load in a mouse model of CMV infection.

Discussion
In this study, we demonstrated that ELP-P10 effectively inhibits the growth of MCMV in cell culture and in an in vivo model of CMV infection.A previous study from our lab has established that the CR2 region of HCMV tegument protein pp150 is amenable to targeting by sequence-specific peptide P10 23 .We used ELP as a suitable conjugate and carrier system to enhance the bioavailability of the selected peptide for future applications in an in vivo system.
We first sought to determine if the P10 peptide still retains its inhibitory potential when fused to the ELP carrier.As shown in Fig. 2c, ELP-P10 successfully inhibited MCMV growth in cell culture.This demonstrates that using ELP as a carrier did not interfere with the ability of P10 to disrupt viral infection, though it did lower the potency of the inhibitory peptide 23 .Ongoing work is exploring strategies to modify the ELP carrier or increase cellular uptake of the construct to increase potency.Interestingly, ELP itself showed a significant reduction in the virus growth.However, the IC 50 of ELP was ~ three-fold higher compared to ELP-P10, and virus inhibition was observed only with the higher concentrations of ELP (Fig. 2a, b).One potential explanation for the non-specific inhibition by ELP is that the large size of the ELP molecule may hinder the interaction of viral proteins during the assembly of the virus.This interference caused by ELP might result in the non-specific inhibition of the virus growth.Another explanation could be the effects of large doses of ELP on cellular endocytosis/exocytosis processes.However, these hypotheses require further research to confirm their validity and to investigate the potential mechanism underlying the observed inhibitory effects of ELP.
To further strengthen our hypothesis that ELP conjugation of P10 will enhance its plasma life, we performed pharmacokinetic and biodistribution analysis with rhodamine-labeled P10 and ELP-P10.As expected, we observed an increase in the plasma half-life and bioavailability of ELP-P10 compared to the free P10 peptide, as the calculated area under the curve for ELP-P10 was five-fold higher compared to P10.Moreover, there was an increased accumulation of ELP-P10 in the mouse organs, specifically in the liver and kidneys.Higher accumulation of ELP-P10 in the liver and kidneys could be attributed to the unique properties of the ELP system.ELPs have an increased half-life and tissue accumulation compared to small peptides which are rapidly filtered by kidneys and cleared from the body more quickly.Due to the larger size of ELPs, their renal filtration is slower than that of free peptides, and they exchange between plasma and tissues more slowly.Therefore ELP-fused peptides accumulate for longer periods of time and at higher levels in organs like the liver and kidneys 30 .This contrasts with the free peptide P10 which is rapidly filtered by the kidneys and thus accumulates in kidneys to a very low level.Additionally, enhanced accumulation of ELP-P10 in the liver could be due to uptake by the reticuloendothelial system.
Further, to investigate if the enhanced bioavailability of ELP-P10 translated to enhanced antiviral efficacy, we tested the ability of ELP-P10 to inhibit virus growth in an in vivo murine model of MCMV infection.The observed percentage change in body weight in the ELP-P10 treated group was less compared to the untreated control group.This suggests a protective effect of ELP-P10 treatment.Moreover, the vehicle-treated (PBS) animals had severe signs of disease such as ruffled fur and hunched behavior compared to the P10, ELP, and ELP-P10 treatment groups.Previous studies on MCMV using a mouse model have shown that intraperitoneal administration of MCMV can cause significant pathology to the host and leads to high viral load in major organs, with maximum viral load in the liver and spleen as these are considered the principal sites of early viral replication [38][39][40] .Similarly, we observed high viral load in the liver and spleen of the vehicle treated mice.Notably, the viral titers from all infected vital organs were reduced by 80-90% in the ELP-P10 treated group, compared with less dramatic reductions in the P10 and ELP control groups.This could be attributed to the fact that ELP conjugation of P10 enhanced its plasma half-life and tissue accumulation.This can result in a more sustained concentration of the therapeutic in the infected organs, which can potentially lead to more inhibition and antiviral efficacy against MCMV.Despite the high viral load observed in the liver of infected mice (vehicle treatment), previous studies have shown that the virus production in liver does not appear to contribute to systemic viral spread 41,42 .CMV is a major opportunistic infection in the liver and kidneys and causes hepatitis and major problems in kidneys of transplant patients 37 .Therefore, the accumulation of the therapeutic in these organs suggests its potential to be used in transplant settings to reduce CMV infection.Altogether, our findings represent an interesting proof of principle in demonstrating the inhibition of MCMV growth by a peptide targeting a non-surface viral protein.While ELP-P10 holds the potential to be developed into an effective therapeutic against cytomegalovirus infection, it is crucial to acknowledge that the calculated IC 50 of ELP-P10 is several log steps higher than the approved antivirals for use in clinical applications.For instance, Enfuvirtide-an FDA approved antiviral for the treatment of HIV, has IC 50 in the nanomolar range; Myrcludex B for treatment of Hepatitis B and D has demonstrated efficacy in picomolar concentrations 43,44 These antivirals underscore the significance of achieving antiviral activity at lower dosage.Of note, the IC 50 of unconjugated P10 is 1.3 µM, however, on conjugation with ELP there is almost a 20-fold increase in the IC 50 23 .While a high IC 50 is a concern, the IC 50 is not the only parameter that determines the clinical viability of a novel therapeutic.The potential advantages conferred by ELP modification, such as prolonged plasma half-life and increased accumulation in the organs, plus the ease of producing ELP-P10 in gram quantities by recombinant production methods, may outweigh the cost of a higher IC 50 of ELP-P10.Overall, our study indicates that ELP-P10 inhibits MCMV growth in vitro and in vivo.Therefore, it can serve as a potential antiviral for combating CMV infection.

Virus
MEFs were used to culture the K181 strain of MCMV.To prepare the virus stock, the MCMV was mixed with 3× autoclaved milk, which was derived from Carnation (Nestle) instant nonfat dry milk powder.The mixture was sonicated three times for 10 s each, with a 30-s gap in between, and then stored at − 80 °C.Additionally, 10% milk was prepared in nanopure water, with a pH of 7.0, and autoclaved three times.

Generation of the ELP-P10 construct
A synthetic DNA cassette encoding the P10 sequence was synthesized with codons that were optimized for expression in E. coli (Life Technologies).The cassette was inserted into the pET25b+ expression vector between NdeI and BamHI restriction sites, with an SfiI site at the N-terminus of the P10 coding sequence.DNA containing

Cell viability
MEFs were plated in 24-well tissue-culture plates and grown to confluency in triplicates.Cells were pretreated for 1 h with 80 μM ELP and ELP-P10, and GCV (control) or vehicle-treated, then infected with MCMV at an MOI of 3.0 or mock-infected.At 3 dpi, the CMC (carboxymethylcellulose) + DMEM overlay was removed.The cells were then harvested by trypsinization to determine the cell viability using trypan blue exclusion with a TC20 automated cell counter (BioRad Laboratories, Hercules, CA, USA) following the manufacturer's protocol.Cell viability was determined using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS).Breifly, MEFs were plated in 96 well plate and allowed to grow for 24 h followed by pretreatment with desired concentrations of ELP, ELP-P10 and GCV for 1 h, and then infected with MCMV at MOI of 3.0 or mock-infected.The cells were washed twice with PBS and overlayed with CMC + DMEM and incubated for 3 days.At 3 dpi, the CMC + DMEM overlay was removed, followed by addition of Complete DMEM and MTS reagent in a ratio of 5:1.The plates were incubated at 37 °C with 5% CO 2 for 1-1.5 h.Absorbance was measured at 490 nm using Tecan's Magellan Microplate Reader.The percentage of cell survival was plotted for different treatment groups, and data were analyzed by one-way ANOVA using Tukey post hoc test.

Rhodamine labeling of ELP-P10
ELP-P10 was fluorescently labeled on a unique cysteine residue using tetramethylrhodamine-5-maleimide.ELP-P10 was diluted to 100 µM in sterile PBS.The diluted protein was then mixed with 1 mM TCEP (tris-(2-carboxyethyl) phosphine) in Na 2 H 2 PO 4 buffer for 1 h while rotating at room temperature followed by the addition of rhodamine to a final concentration of 200 μM.This mix was incubated with continuous stirring overnight at room temperature to ensure the labeling of protein with rhodamine.Any remaining free dye was removed by thermal cycling.Protein concentration and efficiency of labeling on the single cysteine residue were assessed by UV-Visible spectrophotometry as previously described 45 .

Mice
All animal studies were conducted in accordance with the ARRIVE guidelines and the protocols approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center.All experiments were performed in accordance with relevant guidelines and regulations.Male and female Balb/c mice (6-9 weeks of age), maintained under specific pathogen-free conditions, were used for this study.

Pharmacokinetics and biodistribution analysis of peptides
Mice (female, n = 4 per group) were anesthetized with isoflurane and administered 500 nmol/Kg of rhodaminelabeled ELP-P10/P10 by subcutaneous injection.Blood samples were collected from the tail vein at different time www.nature.com/scientificreports/points and centrifuged to collect plasma.The fluorescence of plasma samples was directly measured using a Take 3 Microvolume Plate, measured using an Agilent Cytation 7 (Excitation: 535, Emission: 580, Gain: 100), and fit to a standard curve of rhodamine-labeled ELP and ELP-P10.For the biodistribution, a separate cohort of mice (female, n = 4 per group) were euthanized four hours after the injection with proteins or saline control, and the brain, spleen, liver, lungs, kidneys, and heart were harvested and assessed by ex-vivo whole organ imaging using the In Vivo Imaging System (IVIS, Perkin Elmer).The average radiant efficiency of each organ was adjusted by subtracting the autofluorescence values obtained from imaging the organs of an animal injected with saline and fit to a standard curve of known concentrations of ELP or ELP-P10.To determine pharmacokinetic parameters, plasma concentrations during the clearance phase (from the peak of the plasma concentration curve) were fit using a two-compartment pharmacokinetic model as described previously using GraphPad Prism 32 .

In-vivo analysis of peptide efficacy
Mice (3 males, 3 females per treatment group) were anesthetized with isoflurane and subcutaneously injected with 1000 nmol/Kg of the P10, ELP, ELP-P10, or vehicle-treated 1 h prior to infection.One-hour post-treatment, mice were infected intraperitoneally with 1 × 10 6 PFU of MCMV K181.Animals were treated with the same dose of peptides every 24 h, and their weight was monitored.Three days post-infection, animals were euthanized, and the liver, kidneys, spleen, heart were harvested and stored at − 80 °C.Virus titers in harvested organs were determined by plaque assay.

Viral titers by plaque assay
MEFs were used for the plaque assays.MCMV-infected samples were harvested and stored at − 80 °C.The harvested organs were thawed at 37 °C and then stored on ice.The organs were then sonicated in PBS three times for 10 s each with a 10-s gap.Monolayers of MEFs were grown in 12 well tissue culture plates, and serial dilutions (10 -1 to 10 -3 ) of sonicated samples were absorbed on them for 2 h at 37 °C in 5% CO 2 in triplicates.After 2 h, the sonicate was removed by suction followed by two washes with PBS.Then, pre-warmed CMC dissolved in complete DMEM was added and incubated for 3 days.At the endpoint, the CMC media was removed, cells were washed 2 times with PBS and fixed with 100% methanol for 7 min.Finally, the monolayers were stained with crystal violet for 30 min, then washed with tap water, air dried, and MCMV plaques were quantified.

Statistics
Results from all experiments are expressed as mean ± SD or otherwise stated.Comparisons among three or more groups were made using one-way ANOVA or two-way ANOVA, as appropriate, in GraphPad Prism 9.0 (GraphPad Prism version 9.0.0,GraphPad Software, San Diego, CA, SA, access date 10 November 2021) with Tukey's post-hoc tests (comparing mean of treatment groups to the vehicle) to correct for multiple comparisons.Differences between or among groups were considered significant at a p value of < 0.05.

Figure 1 .Figure 2 .
Figure 1.Illustration of the peptide constructs: the unconjugated P10 peptide (top) and ELP-conjugated P10 peptide (bottom) with the linker sequences shown in grey.ELP-P10 chimeric fusion construct was constructed by conjugating the P10 peptide to the C terminal of ELP.

Figure 3 .
Figure 3. Cell Viability (%) in (a) infected MEFs (b) uninfected MEFs with different treatments groups.MEFswere pretreated with ELP and ELP-P10, and GCV (control) or vehicle-treated for 1 h in triplicates, and then infected with a high MOI of 3.0 with MCMV K181 or vehicle-infected.Cells were harvested at 3 dpi, and cell viability was assessed by trypan blue exclusion assay for both uninfected and infected groups.The standard error of the mean from three different experiments were plotted as error bars.Data were analyzed in GraphPad Prism with one-way ANOVA and showed significant differences (p < 0.05) between groups.

Figure 4 .
Figure 4. Pharmacokinetics and biodistribution of P10 and ELP-P10.Rhodamine-labeled P10 (free peptide) or ELP-P10 were administered by subcutaneous injection in mice (females, n = 4 per group).Plasma pharmacokinetics (a) were determined for 72 h after injection.In a separate cohort of mice (females, n = 4 per group), major organ biodistribution was determined 4 h after injection by ex vivo whole-organ fluorescence imaging (b and c).The standard deviations were plotted as error bars.

Figure 5 .
Figure 5. Efficacy of P10 (free peptide) and ELP-P10 in MCMV-infected mice: (a) Schematic of experimental setup.Mice (BALB/c, 3 males and 3 females per treatment group) were administered with 1000 nmol/Kg of P10 or ELP or ELP-P10 or vehicle subcutaneously followed by intraperitoneal infection with 10 6 pfu/mL of MCMV (K181 strain) one-hour post treatment.Mice were sacrificed on day 4 post-infection and vital organs were harvested for titering.(b) Percentage change in body weight of animals with different treatment groups.Viral load in MCMV infected mouse organs (c) Liver, (d) Kidneys, (e) Spleen and (f) Heart with different treatments assessed by plaque assay.Data were analyzed in GraphPad Prism with one-way ANOVA and post-hoc Tukey's multiple comparisons, asterisks represent p-value < 0.05.The standard deviations were plotted as error bars. https://doi.org/10.1038/s41598-024-60691-6